In studies of tumour development, various in-vitro models have been applied. Generally, cells are grown in two-dimensional monolayers on plastic plates covered with liquid media that supplies essential nutrients and growth factors for the cells.
Even though two-dimensional monolayer culture has provided great benefits to scientists and clinicians, this culture system suffers from a particular lingering disadvantage. This method of cell culture does not mimic effectively the in-vivo environment from which the cells were originally isolated. Cells, such as tumour cells, do not grow in two-dimensional monolayers within the body. Rather, the in vivo environment involves interactions between cells of different types in three dimensions. Thus, two-dimensional monolayer or two-dimensional suspension cell cultures cannot accurately reflect the true three-dimensional cellular architecture found in vivo.
Unsurprisingly, cells cultured in monolayers do not exhibit the same biological responses seen in vivo. In a monolayer, all of the cells have the same growth conditions which results in a homogenous cell population wherein every cell is like every other cell in the culture system. In contrast, naturally occurring cells generally represent a heterogeneous cell population resulting, for example, from positional cues, cell differentiation and differences in the multi-cellular and biochemical environment such as hormones, growth factors, oxygen tension, cytokines, chemokines etc.
To mimic the properties of the naturally occurring cellular environment more closely, three-dimensional cell culture systems have been developed for use in medical and biological research. Usually these systems utilise well established cell lines on the basis that their use allows standardisation and comparability of results between experiments. These three-dimensional cultures are essentially homotypic, meaning that they are made up of only one cell type. Thus, such cultures cannot reflect accurately the heterotypic in vivo environment.
Fibroblasts have key functional roles in the tissues in which they reside, synthesizing and maintaining the extracellular matrix of body tissue. Fibroblasts provide a structural framework (stroma) for many tissues, play a critical role in wound healing and are the most common cells of connective tissue in animals.
The main function of fibroblasts is to maintain the structural integrity of connective tissues by continuously secreting precursors of the extracellular matrix. Fibroblasts secrete the precursors of all the components of the extracellular matrix, primarily the ground substance and a variety of fibres. The composition of the extracellular matrix determines the physical properties of connective tissues.
Further, fibroblasts are capable of producing cytokines (such as e.g. interleukins, cgfbeta, IGF-1), chemokines (such as e.g. CXCL12), growth factors (such as e.g. hgf, vegf, fgf, egf), proteases (such as e.g. MMPs, CIMPs) and other soluble factors (such as e.g. S100A4).
Fibroblasts are morphologically heterogeneous with diverse appearances depending on their location and activity. Ectopically transplanted fibroblasts often retain positional memory of the location and tissue context where they had previously resided, even over several generations.
Unlike epithelial cells that line the bodies structures, fibroblasts do not form flat monolayers and are therefore not restricted by a polarising attachment to a basal lamina on one side. Fibroblasts can also migrate slowly over the substratum as individual cells. Whilst epithelial cells, for example, form the lining of body structures, fibroblasts and related connective tissues sculpt the “bulk” of an organism.
As a result of these interactions with other cells, fibroblasts have been found to play a role in tumour formation. As tumour fibroblasts or myo-fibroblasts, they are believed to be important in both tumour development and tumour progression.
For these reasons, heterotypic spheroids have been developed as three dimensional tumour models wherein standardised cell lines are combined with fibroblasts. These methods require large amounts of fibroblast cells. Unfortunately, fibroblasts can so far only be grown in the laboratory in small quantities.
Further, to generate spheroids closely resembling the natural tumour and/or tissue characteristics, the fibroblasts used have to be of a heterogenic type.
Thus, there is a need in the art for methods of producing large amounts of fibroblasts, particularly from limited amounts of starting material which are of heterogenic type.